This project has as a long range goal the development of methods to modulate opioid peptide action through the peptidases which inactivate them. A major focus of this research is the enzyme neutral 24.11 (NEP, enkephalinase) which is involved in the inactivation of endogenously released enkephalins. We propose to identify the active site of NEP by chemical modification experiments in conjunction with the analysis of recombinant enzyme modified by site-directed mutagenesis. Peptides containing the photo activatable azido group will be used to locate amino acids in specific binding subsites with mutagenesis used to delineate the role of these residues. The functionality of an active site arginine will be studied by converting this arginine to lysine, glutamate, and glycine. These mutant enzymes will be used to evaluate the contribution of this arginine to catalytic efficiency and as a determinant of substrate specificity. The enzyme has been shown to catalyze hydrolysis at structured residues within cyclic peptides. We will therefore study the effect of peptide structure on the specificity of the enzyme. Irreversible inhibitors for NEP will be prepared based on the structure of N-bromoacetyl-D-Leu-Gly, a compound we recently synthesized as the first irreversible NEP inhibitor. These inhibitors will be used to study the role of NEP in the metabolism of other opioid and non-opioid peptides. A major goal of this project is to generate a crystalline form of NEP suitable for X-ray diffraction analysis. Several recombinant expression systems will be explored for the generation of high levels of wild type and mutant NEP. Studies are also proposed relating to the puromycin sensitive aminopeptidase. A membrane bound form of this enzyme was shown to be unique to brain. A cDNA for the enzyme will be isolated and used to determine if a brain specific mRNA exists and whether or not the levels of this mRNA are modulated in response to opiates. The active site of the enzyme will be identified through chemical modification using azido phenylalanine derivative. Site directed mutagenesis will be used to study the functionality of identified residues. We propose to identify, purify, and characterize peptidases which may regulate beta-endorphin action by degradation. Synthetic peptides will be used to identify the peptidases active on beta-endorphin and as an assay during their purification. Monoclonal antibodies will be generated against each of the peptidases and used as metabolic inhibitors. These studies should lead to the synthesis of specific inhibitors, which like the effect of NEP inhibitors on endogenous enkephalins, may be able to modulate beta-endorphin levels in vivo.